2-ethylhexyl cyanoacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl cyanoacetate

EC Number:

236-425-5

EC Name:

2-ethylhexyl cyanoacetate

Cas Number:

13361-34-7

Molecular formula:

C11H19NO2

IUPAC Name:

2-ethylhexyl 2-cyanoacetate

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

TA 98: hisD3052, rfa, uvrB
TA 100: hisG46, rfa, uvrB
TA 102: hisG428, rfa
TA 1535: hisG46, rfa, uvrB
TA 1537: hisC3076, rfa, uvrB

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat)

Test concentrations with justification for top dose:

25, 50, 80, 160 and 250 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without metabolic activation

Migrated to IUCLID6: 2.5 µg/plate

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without metabolic activation

Migrated to IUCLID6: 5.0 µg/plate

Controls 4

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA 102 without metabolic activation

Migrated to IUCLID6: 2.5 µg/plate

Controls 5

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without metabolic activation

Migrated to IUCLID6: 40.0 µg/plate

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-Aminofluorene 100 µg/plate, 2-Aminoanthracene 2.5 µg/plate

Remarks:

TA 1535, TA 1537, TA 98, TA 100 and TA 102 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st main experiment: in agar (plate incorporation); 2nd main experiment: preincubation

DURATION
- 1st main experiment: 72 hours incubation (37°C)
- 2nd main experiment: 30 min preincubation (30°C), 72 hours incubation (37°C)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: determination of background lawn

Evaluation criteria:

Validity criteria:
- In the solvent control, each tester strain culture must exhibit a characteristic mean number of spontaneous revertants
- To ensure that appropriate numbers of bacteria are plated, overnight culture titers must be in excess of 108 bacteria/mL.
- The mean of each positive control must exhibit a significant increase in the number of revertants over the mean value of the respective vehicle control. In case of no significant increase in the number of revertants by addition of 2-Aminofluorene, a parallel significant increase by addition of 2-Aminoanthracene will be regarded as sufficient and vice versa.
- Normally, at least four non-toxic dose levels are required to evaluate the assay data. Exceptions from this requirement, however, may be justified.

Criteria for positive response:
For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect. Exceptions from this requirement, however, may be justified.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under conditions tested, the test item did not induce gene mutations by base pair changes or frame-shifts in the genome of the tester strains used.
Thus, the test item is considered non-mutagenic in this bacterial reverse mutation assay.